Process of inhibiting cell death in injured cartilage

ABSTRACT

Processes for inhibiting apoptotic cell death and glycosaminoglycan release from injured cartilage is provided. Inhibition is accomplished using caspase inhibitors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/099,408, filed Mar. 15, 2002 (now pending), which claims priority toU.S. Provisional Patent Application Ser. No. 60/276,183, filed Mar. 15,2001 (now expired). The full disclosures of the earlier filedapplications are incorporated herein by reference in their entirety andfor all purposes.

Funds used to support some of the studies reported herein were providedby the United States Government (NIH Grant AG07996). The United StatesGovernment, therefore, has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

The field of this invention is cartilage injury. More particularly, thepresent invention pertains to a process for inhibiting apoptotic celldeath and glycosaminoglycan release in mechanically injured cartilageusing caspase inhibitors.

BACKGROUND OF THE INVENTION

Articular cartilage has poor reparative potential, but the reasons forthis are not fully understood (Archer (1994) Ann. Rheum. Dis. 53,624-630, Silver, et al. (1993) Otolaryngol. Clin. North Am. 28,847-864). The lack of tissue vascularization and the putative absence ofstem cells are potential explanations. Although histologic studiesdemonstrate the occurrence of chondrocyte death in response tomechanical injury, it is not fully known which stimuli trigger celldeath and whether cell death occurs as apoptosis or necrosis(Calandruccio, et al. (1962) J. Bone Joint Surg. Am. 44A, 431-455,Mankin (1962) J. Bone Joint Surg. Am. 44A, 682-688, Bentley, et al.(1971) Nature 230, 385-388, Repo, et al. (1977) J. Bone Joint Surg. Am.59A, 1068-1076). Cartilage, unlike other tissues, has no means ofremoving dead cells by phagocytosis due to the absence of tissuemacrophages. Consequently, it may be proposed that lesions containingapoptotic or necrotic cells are detrimental, partly explaining poorintegration with surrounding articular cartilage which is a commonfeature of most reported repair models. However, where this zone of celldeath was resorbed by added macrophages, full repair has been reported(Joseph, et al. (1961) J. Anat. 95, 564-568).

Similarly, in marginal regions of injured meniscus where cell death wasnot observed, repair can be complete (Walmsley, et al. (1938) J. Anat.12, 260-263). Collectively, these findings suggest that chondrocytedeath may be one of the limiting factors in the response of cartilage toinjury. However, information on the induction of cell death in responseto mechanical injury is limited. It has recently been proposed that celldeath in response to wounding is a combination of necrosis and apoptosis(Tew, et al. (2000) Arthritis Rheum. 43, 215-225). This distinction maybe critical since apoptosis can be inhibited resulting in a potentialincrease in cell viability. Apoptosis has been inhibited in varioussettings (Rudel (1999) Herz. 24, 236-241).

Joint loading and motion can induce a wide range of metabolic responsesin cartilage. Immobilization or reduced loading leads to a decrease inglycosaminoglycan (GAG) synthesis and content (Caterson, et al. (1978)Biochim. Biophys. Acta. 540, 412-422, Kiviranta, et al. (1987) ArthritisRheum. 30, 801-809, Guilak (1994) J. Microsc. 173, 245-256, Sah, et al.(1989) J. Orthop. Res. 7, 619-636, Burton-Wurster, et al. (1993) J.Orthop. Res. 11, 717-729, Kim, et al. (1994) Arch. Biochem. Biophys.311, 1-12). Increased dynamic loading causes an increase in GAGsynthesis and content (Caterson, et al. (1978) Biochim. Biophys. Acta.540, 412-422, Kirviranta, et al. (1987) Arthritis Rheum. 30, 801-809,Sah, et al. (1989) J. Orthop. Res. 7, 619-636, Gray, et al. (1988) J.Orthop. Res. 6, 777-792, Jones, et al. (1982) Clin. Orthop. 165,283-289, Sah, et al. (1991) Arch. Biochem. Biophys. 286, 20-29). Moresevere static or impact loading causes cartilage deterioration and leadsto osteoarthritic changes (Repo, et al. (1977) J. Bone Joint Surg. Am.58A, 1068-1076, Gritzka, et al., J. Bone Joint Surg. Am. 55A, 1698-1720,Radin, et al. (1984) J. Orthop Res. 2, 221-234, Thompson, et al. (1991)J. Bone Joint Surg. Am. 73A, 990-1001). In fact, traumatic cartilageinjury represents a major risk factor for the development of secondaryosteoarthritis.

Prior histologic studies have demonstrated the occurrence of cell deathafter articular cartilage injury (Calandruccio, et al. (1962) J. BoneJoint Surg. Am. 44A, 431-455, Mankin (1962) J. Bone Joint Surg. Am. 44A,682-688). More recently, cell death in response to articular cartilagewounding has been reported (Tew, et al. (2000) Arthritis Rheum. 43,215-225). Electron microscopy and TUNEL evidence of both necrosis andapoptosis was seen in a band along the would margins. An increase in theband of cell death was observed over the first 5 days following thewounding. These phenomena were demonstrated in explants from bovinemetacarpal and metatarsal joints with the production of a manuallycreated cartilage defect. Clinically, accidental blunt trauma is by farthe more common form of injury leading to cartilage lesions. Articularcartilage can sustain injury without apparent loss of matrix. It ispossible that cell death (whether apoptotic or necrotic) may occurgiving rise to later matrix degradation and the subsequent developmentof a full thickness cartilage lesion. Another recent study by Loening etal. demonstrated apoptosis after a similar injurious loading ofcartilage explants (Loening, et al. (2000) Arch. Biochem. Biophys. 381,205-212). Recently, broad spectrum caspase inhibitors and selectivenon-peptide caspase inhibitors have been successfully used to inhibitapoptosis induced by several agents in cultured human chondrocytes (Lee,et al. (2000) J. Biol. Chem. 275, 16007-16014, Nuttall, et al., (2000)J. Orthop. Res. 18, 356-363). In the setting of mature articularcartilage with a limited source of chondrocytes, maintaining viabilitycould substantially impact subsequent degeneration and repair.

The experimental models used in most prior studies are not especiallyuseful in predicting clinical outcomes. There is a need therefore forclinically relevant studies disclosing the mechanisms underlying celldeath in injured cartilage. The model used in the current study waschosen to represent a type of blunt trauma that is more clinicallyrelevant.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process of inhibiting apoptotic celldeath in cartilage following mechanical injury. The process includes thestep of inhibiting the activity of cysteine-aspartate-specific proteasesin the injured cartilage. Preferably, the activity ofcysteine-aspartate-specific proteases is inhibited by contacting thecartilage with an inhibitor of cysteine-aspartate-specific proteases. Anespecially preferred such inhibitor is a broad based inhibitor ofcysteine-aspartate-specific proteases. One especially preferred suchinhibitor is a fluoromethylketone caspase inhibitor such asbenzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. In anotheraspect, this invention provides a process of inhibitingglycosaminoglycan (GAG) release from cartilage following mechanicalinjury of the cartilage, wherein the process includes the step ofinhibiting apoptotic cell death in the injured cartilage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides processes for inhibiting apoptotic celldeath and GAG release from cartilage following mechanical injury. Theprocesses include the step of inhibiting, in the injured cartilage, theactivity of cysteine-aspartate-specific proteases. Apoptosis is mediatedby a cascade of aspartate-specific cysteine proteases or caspases (Rudel(1999) Herz. 24, 236-241). The family of cysteine aspartate-specificproteases (caspases) are mediators of cell death. Caspases cleavestructural proteins, and regulators of transcription, DNA replication,DNA/RNA metabolism, and DNA degradation (Nuttall, et al. (2000) J.Orthop. Res. 18, 356-363, Elkon (1999) J. Exp. Med. 190, 1725-1728).Caspase-8 has been shown to play a central role in signal transductiondownstream of cell membrane death receptors (Martin, et al. (1998) J.Biol. Chem. 273, 4345-4349, Muzio, et al. (1998) J. Biol. Chem. 273,2926-2930). In this pathway, caspase-8 activates caspases-3 and -7,while caspase-3 activates caspase-6. Caspases-3 and -6 cleave proteinsleading to nuclear apoptosis (Hirata, et al., (1998) J. Exp. Med. 187,587-600). Mitochondria have also been implicated in apoptosis. One ofthe first events detected is a drop in the mitochondrial transportmembrane potential. This is associated with release of cytochrome c intothe cytoplasm which results in activation of caspase-9 (Kroemer (1999)Biochem. Soc. Symp. 66, 1-15, Kuida (2000) Int. J. Biochem. Cell Biol.32, 121-124). More recently, caspase-12 has been implicated in apoptosisresulting from stress in the endoplasmic reticulum (Nakagawa, et al.(2000) Nature 403, 98-103). Caspases therefore appear to be thedownstream executors in almost all forms of apoptosis. In accordancewith the present invention, therefore, cysteine-aspartate-specificprotease activity is preferably inhibited through the use of caspaseinhibitors, as are well known in the art. Exemplary caspase inhibitorsare shown in Table 1, below. Broad spectrum caspase inhibition can beaccomplished using a variety of fluormethylketones. An exemplary andpreferred such fluormethylketone is benzyloxycarbonyl-Val-Ala-Asp-(OMe)fluoromethyl ketone.

TABLE 1 Caspase-1 Inhibitor: YVAD-FMK Caspase-2 Inhibitor: VDVAD-FMKCaspase-3 Inhibitor: DEVD-FMK Caspase-4 Inhibitor: LEVD-FMK Caspase-5Inhibitor: WEHD-FMK Caspase-6 Inhibitor: VEID-FMK Caspase-8 Inhibitor:IETD-FMK Caspase-9 Inhibitor: LEHD-FMK Caspase-10 Inhibitor: AEVD-FMKCaspase-13 Inhibitor: LEED-FMK Y = Tyr V = Val A = Ala D = Asp E = Glu L= Leu W = Trp I = Ile T = Thr H = His

Inhibition of caspase activity in cartilage is accomplished by exposingthe cartilage to an effective caspase inhibitory amount of theinhibitor. Exposing is accomplished by providing an effective amount ofthe inhibitor in fluid bathing or perfusing the cartilage. Where thecartilage is situated in vitro or in situ, the effective amount of theinhibitor is added to the medium bathing or surrounding the cartilage.The cartilage is then maintained in that medium for a period of timesufficient for caspase inhibition. Suitable media for maintainingcartilage in vitro or in situ are well known in the art.

There is dose response relationship between the amount of inhibitor andthe inhibition of apoptotic cell death and GAG release. Thus, apreferred effective amount of a caspase inhibitor is from about 20 μm toabout 250 μm. More preferably, the inhibitor is present in an amount offrom about 50 μm to about 150 μm. Where the cartilage is situated invivo, the inhibitor is systemically administered as to achieve effectivelevels in the blood and interstitial fluid perfusing the cartilage. Oneof skill in the art can readily calculate the amount of inhibitor neededto achieve such levels.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a caspaseinhibitor in combination with a pharmaceutically acceptable carrier. Asused herein, the terms “pharmaceutically acceptable”, “physiologicallytolerable” and grammatical variations thereof, as they refer tocompositions, carriers, diluents and reagents, are used interchangeableand represent that the materials are capable of administration to orupon a human without the production of undesirable physiological effectssuch as nausea, dizziness, gastric upset and the like which would be toa degree that would prohibit administration of the composition.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified. The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, as well as pHbuffering agents and the like which enhance the effectiveness of theactive ingredient.

The therapeutic pharmaceutical composition of the present invention caninclude pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol and other solutes. Liquid compositions can alsocontain liquid phases in addition to and to the exclusion of water.Exemplary of such additional liquid phases are glycerin, vegetable oilssuch as cottonseed oil, organic esters such as ethyl oleate, andwater-oil emulsions.

Cartilage explants obtained from bovine and human bone were subjected toa variety of mechanical load injuries (See the Examples hereinafter).The release of GAG from injured cartilage and the extent of apoptosis inthe injured cartilage was determined. GAG release was measured todetermine if this was dose related and occurred in a time dependentmanner.

In bovine cartilage, GAG release (expressed as a percent of total GAGcontent of the explant) increased with intensity of loading at 48 hoursafter loading. There was no significant difference between GAG releaseof Control (mean 1%, SD±0.7) and Low Load explants (mean 3%, SD±0.8).However, both the Moderate Load (mean 4%, SD±0.3) and High Load (mean19%, SD±18.9) explants demonstrated a higher level of GAG releasecompared with Control (p<0.02). A few explants in the High Load groupsuffered structural damage and released much higher levels of GAG thanothers. When analyzed separately (undamaged vs. damaged), the HighLoad-damaged group demonstrated significantly higher GAG release ratesthan High Load-undamaged group (p<0.01). In the time course series, GAGrelease was consistently higher for the loaded explants at all the timepoints tested.

In human cartilage, GAG release (expressed as a percent of total GAGcontent of control explants) increased significantly with injury at 96hours after loading. Loaded explants (mean 1.9%, SD±0.14) demonstrated ahigher level of GAG release compared with Control (mean 0.8%, SD±0.28)(p<0.02). This was also confirmed by Safranin-O staining of histologicalsections which showed reduced intensity of stain from the upperone-third to one-half of the loaded cartilage sections.

The extent of apoptosis was studied to establish whether chondrocyteapoptosis occurs in response to mechanical injury and to determine theresponse to different load magnitudes. In bovine cartilage, at 48 hours,control explants demonstrated mean baseline apoptosis of 18% (0-30%).Low Load explants did not demonstrate any significant difference at mean15% (0-30%). Both Moderate Load and High Load explants exhibitedsignificantly higher apoptosis rates at 40% (range, 22-48%) and 47%(range, 28-60%) respectively (p<0.01). The time course experimentsdemonstrate that the percentage of apoptotic cells increases in atime-dependent manner up to 48 hours after injury. In addition, acorrelation of GAG release with apoptosis rate was noted (r=0.67,p<0.01). Control explants demonstrated fewer apoptotic cells which werelocated mainly in the superficial zone, while High Load explantsrevealed apoptotic cells in superficial and intermediate zones with veryfew in the deep zone. Explants treated with sodium nitroprussidedemonstrated a widespread pattern of apoptosis. Confirmation thatchondrocytes were indeed undergoing apoptosis was obtained by electronmicroscopy. No significant increase in the incidence of apoptosis wasseen at the cut edges of the explants between control and loadedexplants.

In human cartilage, explants were examined by TUNEL to determine ifchondrocyte apoptosis does occur in response to mechanical injury. At 96hours, Control explants demonstrated mean baseline apoptosis of 4%(SD±2). Loaded implants (stress level=14 MPa) exhibited significantlyhigher apoptosis rates, mean 34% (SD±11, p<0.01). Control explantsdemonstrated a few apoptotic cells which were located mainly in thesuperficial zone, while loaded explants revealed apoptotic cells insuperficial and intermediate zones. The deep zones consistentlydemonstrated minimal or no apoptosis. Electron microscopy revealedapoptotic chondrocytes in the loaded explants but none in the controlexplants. Immunohistochemical staining with the M30 monoclonal antibodyalso confirmed results obtained by TUNEL in the samples tested. Nosignificant increase in the incidence of apoptosis was seen at the cutedges of the explants in either control or loaded explants suggestingthat the explant harvesting procedure did not induce significantapoptosis. In both bovine and human cartilage, there was a significantcorrelation of GAG release with apoptosis rate.

Apoptosis is known to occur after an ordered sequence of cellularevents. Although apoptosis can be induced by several different triggers,it is characteristically associated with the sequential activation ofcaspases. The time course of apoptosis is therefore of particularsignificance as it would determine the presence or absence of atherapeutic window for potential modulation of the apoptotic process.Two series of experiments were performed to determine the time course ofapoptosis after mechanical injury. The first set of experimentsdemonstrated no significant increase in the percentage of apoptoticcells up to 6 hours after injury. The percentage of cells undergoingapoptosis increased from 6 hours after injury to 96 hours after injury.In the second set of experiments, wider time intervals were used and thepercentage of apoptosis was found to continue to increase up to 7 daysafter injury.

The results demonstrate that cartilage mechanical injury in vitro canresult in chondrocyte apoptosis within the range of load intensitiestested. Apoptosis appears to be dose related and increases withincreasing loads. The percentage of cells undergoing apoptosis wasmeasured by TUNEL and confirmed by electron microscopy. The distributionof TUNEL positive cells was consistent within groups. Control explantsdemonstrated a few scattered apoptotic cells in the superficial zone,while higher load explants showed more apoptotic cells in thesuperficial and intermediate zones, with very few in the deeper zones.This increase in the percentage of apoptotic cells found close to thearticular surface shows that superficial cells are more susceptible thandeeper cells.

The demonstration of load induced apoptosis in cartilage also hasimplications for the pharmacologic modulation of post-traumaticcartilage lesions. The present study also demonstrated that caspaseinhibition reduces chondrocyte apoptosis after mechanical injury. Thus,following mechanical injury to cartilage, there is likely a time periodduring which chondrocyte apoptosis is sensitive to pharmacologicinhibition. This has not been observed in a previous report of cartilageinjury (Tew, et al. (2000) Arthritis Rheum. 43, 215-225).

Proteoglycan release has previously been used as a measure to estimatethe extent of mechanical injury. In the present study there was a cleardose dependent release of proteoglycan in response to the intensity ofload. This validates that in vitro loading (at 14 and 23 MPa) produces areproducible metabolic response indicative of cartilage injury. Quinn etal. reported an increase in cell-mediated matrix catabolic processeswith increased rates of proteoglycan turnover following cartilage injurythat may explain the increased loss of GAG in media (Quinn, et al.(1999) Ann. N.Y. Acad. Sci. 878, 420-441). More recently, Lee et al.,reported that apoptosis prevention restored type II collagen promoteractivity in chondrocytes (Lee, et al. (2000) J. Biol. Chem. 275,16007-16014). In the present study, inhibition of chondrocyte apoptosiswas associated with a reduction in proteoglycan depletion. This showsthat cell death even in the form of apoptosis is likely linked to matrixloss in cartilage. Both apoptosis and proteoglycan release correlatedwith load intensity and with each other, suggesting a possible linkbetween apoptosis and matrix loss after mechanical injury.

The contribution of cell death to cartilage degradation has beenpreviously suggested for human and experimentally induced osteoarthritis(Hashimoto, et al. (1998) Proc. Natl. Acad. Sci. USA 95, 3094-3099,Hashimoto, et al. (1998) Arthritis Rheum. 41, 1266-1274, Horton, et al.(1998) Matrix Biol. 17, 107-115). In these cartilage pathologies, aclose correlation between the frequency of chondrocyte apoptosis andseverity of osteoarthritic changes was seen. Possible mechanisms are thepresence and release of active enzymes that cause matrix calcificationor degradation from apoptotic bodies (Hashimoto, et al. (1998) Proc.Natl. Acad. Sci. USA 95, 3094-3099). A correlation has been foundbetween the level of nitric oxide production, chondrocyte apoptosis andmatrix depletion in a rabbit model of osteoarthritis (Hashimoto, et al.(1998) Arthritis Rheum. 41, 1266-1274). More recently, Pelletier et al.,reported on a reduction of the severity of experimental canineosteoarthritis, chondrocyte apoptosis, and caspase 3 activity, aftertreatment with a selective inhibitor of inducible nitric oxide syntheses(Pelletier, et al. (2000) Arthritis Rheum. 43, 1290-1299). Theinhibition of apoptosis as a therapeutic modality may therefore have aneven more far reaching impact on osteoarthritis than the initialpreservation of cell viability.

The present disclosure shows that in vitro loading at selected injurylevels, produces reproducible metabolic response indicative of cartilageinjury. More severe static or impact loading causes cartilagedeterioration and leads to osteoarthritic changes. The present resultsalso show that cartilage mechanical injury results in chondrocyteapoptosis at the load intensities used. The percentage of cellsundergoing apoptosis was measured by TUNEL and confirmed using two othermethods: electron microscopy and immunohistochemical staining with M30.Electron microscopy of loaded samples demonstrated cellular patternscharacteristic of apoptosis. An early feature of apoptosis is thecleavage of cytokeratin 18 by caspases. This exposes a neo-epitopespecific for apoptosis which can be detected by M30, a monoclonalantibody. Cells staining positive for this neo-epitope was found mainlyin loaded samples confirming TUNEL results.

Cell death, even in the form of apoptosis appears to be linked to matrixdegradation in cartilage. Since cartilage does not contain tissuemacrophages, there is no apparent mechanism for removing dead cells orapoptotic bodies. This raises the possibility that chondrocyte apoptoticremnants could cause further tissue damage and impact subsequent repair.The present demonstration of a distinct time course of load inducedapoptosis in cartilage therefore has application for the pharmacologicmodulation of posttraumatic cartilage lesions. Results of this studyshow that broad spectrum caspase inhibition can prevent chondrocyteapoptosis in vitro after mechanical injury.

In conclusion, chondrocyte apoptosis can be induced by mechanical injuryin vitro in a dose dependent manner. Apoptosis induced by mechanicalinjury in vitro can be reduced by caspase inhibition. Caspase inhibitionalso reduces the proteoglycan depletion produced by mechanical injury.

Example 1 Bovine Cartilage Studies

Cartilage explants. Macroscopically normal weight bearing portions offreshly slaughtered skeletally mature bovine femoral condyles wereselected from animals between 2 and 6 years of age (Animal Technologies,Austin, Tex.). Only joints without visible signs of degeneration oraging were selected. Using sterile techniques, full thickness cartilagewas separated from underlying subchondral bone with a scalpel and 5 mmdiameter cylindrical explants, ranging from 1 to 1.8 mm in height, coredout using a dermal punch (Acuderm, Inc., Ft. Lauderdale, Fla.). Explantstaken from adjacent sites were used as matched controls for each groupin each experiment to minimize variation in cartilage response due todifferences in thickness and location within the joint. Each explant wasweighed and allowed to stabilize at 37° C. and 5% CO₂ for 48 hours in 1ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%fetal bovine serum (FBS), L-glutamine, antibiotics and 50 μg/ml ascorbicacid.

Full thickness bovine cartilage explants, 5 mm in diameter, weresubjected to a single static mechanical load of 7, 14 or 23 MPa for 500msec. This model simulates traumatic cartilage injury and the loads weresimilar in magnitude to those generated during traumatic joint injury.Glycosaminoglycan (GAG) release and percent apoptotic cells weremeasured. The effect of a pan-caspase inhibitor, z-VAD.fmk[benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone] in preventingchondrocyte apoptosis was determined. z-VAD.fmk is a fluoromethylketoneirreversible inhibitor of a broad spectrum of caspases. A significantincrease in the number of apoptotic cells was observed in response tomechanical loading at 14 and 23 MPa when compared with control. Thepercentage of apoptotic chondrocytes was related to load intensity andcorrelated with the level of GAG release from cartilage. The pan-caspaseinhibitor reduced chondrocyte apoptosis after mechanical injury. Thiswas also associated with a reduction in GAG release. These resultsdemonstrate that mechanical injury induces chondrocyte apoptosis whichis sensitive to pharmacologic inhibition. This identifies a new approachto limit traumatic cartilage injury and the subsequent development ofsecondary osteoarthritis.

Loading apparatus and procedure. A model to simulate acute joint injurywas used in this study based on injury models reported by Repo andFinlay, and Jeffrey et al. (Repo, et al. (1977) J. Bone Joint Surg. Am.59A, 1068-1076, Jeffrey, et al. (1995) Arch. Biochem. Biophys. 322,87-96). Although these reported models impacted cartilage using droptowers, a more controlled method was chosen for applying and maintainingstress or strain as follows. An Instron 8511 servohydraulic testingmachine (Instron Corporation, Boston, Mass.) was used to mechanicallyload the cartilage explants. Explants were transported from theincubator to the Instron machine in sealed cell culture dishes. Theexplant was centralized on a stainless steel loading platform and aradially unconfined compressive load was applied through an impermeablestainless steel platen. A small preload (0.1 MPa) was applied for 2minutes, followed by a single 500 msec trapezoidal loading waveform atthe selected stress. The stress rose from 0.1 MPa to the chosen stresslevel in 100 msec and was maintained for 500 msec. Three stress levelswere chosen to represent acute joint injury. 7 MPa has been reported tobe the upper limit of physiologic stress developed during activities ofdaily living (von Eisenhart, et al. (1999) J. Orthop. Res. 17, 532-539).23 MPa is the stress above which the underlying bone has been reportedto fracture (Haut (1989) J. Orthop. Res. 7, 272-280). Control explantswere placed in the loading apparatus but not loaded. Both control andloaded explants were re-cultured in fresh culture media immediatelyafter loading. Stress and strain data were recorded during the test.

Dose response. To establish extent of tissue response to mechanicalinjury three stress levels were selected: Low Load (7 MPa), ModerateLoad (14 MPa) and High Load (23 MPa). Prior pilot tests demonstratedthat loads below 7 MPa did not result in any measurable cell death andloads above 23 MPa resulted in extensive structural damage of thetissue. The range of maximum tissue compression obtained with eachloading level was mean 40% strain (range 31-49%) for the Low Load group,mean 67% strain (range 44%-77%) for the Moderate Load group, mean 72%strain (range 62-80%) for the High Load (undamaged) and mean 83% strain(range 74-89%) for the High Load (damaged). Four explants each weretested at the three stress levels. Explants taken from adjacentcartilage were selected as paired non-loaded controls (total 12) tocontrol for differences in site of origin.

Glycosaminoglycan release assay. To estimate GAG depletion and release,the concentration of sulfated glycosaminoglycans was measured using1,9-Dimethylene Blue (DMMB) as a monitor of spectrophotometric changeswhich occur during the formation of the sulfated GAG dye complex(Farndale, et al. (1986) Biochim. Biophys. Acts. 883, 173-177, Goldberg,et al. (1993) Agents Actions 39 Spec No, C163-C165). For GAG contentmeasurement, samples were digested in papain and collagenase and theconcentration of sulfated GAG measured using DMMB. To generate astandard curve, chondroitin-6-sulfate (Sigma, St. Louis, Mo.) was usedat concentrations between 1 and 200 μg/ml. GAG released in media wasnormalized to the pooled mean GAG content of Control (uninjured)cartilage explants and expressed as a percentage of total GAG content.

Time response. To establish the time response after injury, control andloaded explants (Moderate Load) were cultured for 2, 4, 8, 24 and 48hours. Percent chondrocytes demonstrating apoptosis and GAG release inmedia were measured at the end of each culture period.

Apoptosis detection. To determine if cell death occurred in the form ofapoptosis in response to mechanical injury, the same three stress levelswere selected: Low Load (7 MPa), Moderate Load (14 MPa) and High Load(23 MPa). Three separate experiments were performed with explants fromsix different bovine joints. In a single experiment, four explants eachwere tested at the three stress levels. Explants taken from adjacentcartilage were selected as paired unloaded controls to control fordifference sin site of origin. Two more explants were treated with 1 mMsodium nitroprusside (SNP) to serve as positive controls for inductionof apoptosis (Blanco, et al. (1995) Am. J. Pathol. 146, 75-85). Explantswere loaded as described above and fixed in 10% buffered formalin 48hours after loading. In situ detection of apoptosis was performed on 5μm thick sections using MEBSTAIN Apoptosis Kit (MBL, Nagoya, Japan).This uses fluorescein-dUTP to label DNA strand breaks (TUNEL method) andhence allows direct detection of DNA fragmentation. TUNEL positive cellsemit a bright green fluorescence while TUNEL negative cells display anorange color due to propidium iodide counterstaining. For each explant,two histologic cross-sections taken completely across the explant wereexamined. Cells in all areas were counted and divided into TUNELpositive or TUNEL negative. TUNEL positive cells seen adjacent to thecut edges of the explant were not included in the analysis since thesewere thought to be due to the surgical trauma at harvest. Sincepropidium iodide would stain all cells in the fixed section, the extentof apoptosis could be quantified by counting the number of TUNELpositive relative to TUNEL negative cells. This was expressed as apercentage of all cells in the explant that exhibited TUNEL positivefluorescence. To serve as a confirmatory test, several control andloaded explants were divided into two halves. One half underwentfluorescein-dUTP labeling as described above. The other half was fixedin 2.5% glutaraldehyde buffered with 0.1 M cacodylate (pH 7.2), rinsedin cacodylate buffer, postfixed for 1 hour in 2% O_(s)O₄ buffered withcacodylate, dehydrated in a graded ethanol series, and embedded inPolybed 812 (Polysciences, Warrington, Pa.). Thin sections were stainedwith uranyl acetate and lead citrate and examined under electronmicroscopy for features of apoptosis.

Inhibition of apoptosis. To determine whether apoptosis could beprevented by caspase inhibition, 12 explants were loaded at 23 MPa (HighLoad) and cultured for 48 hours. For six of these explants, the mediatreated with z-VAD.fmk (100 μM). z-VAD.fmk is a cell-permeablefluoromethylketone peptide inhibitor of caspases and has been shown tobe a broad spectrum apoptosis inhibitor in a variety of cell types(Katsikis, et al. (1997) J. Exp. Med. 186, 1365-1372, Slee, et al.(1996) Biochem. J. 315, 21-24). Six more explants were used asnon-loaded controls. Percent apoptosis and GAG release in media wasmeasured as described above.

Example 2 Human Cartilage Studies

Cartilage explants. Macroscopically normal tibial and femoral articularsurfaces were selected from human cadaver donors between 18 and 45 yearsof age. Using sterile techniques, full thickness cartilage was separatedfrom underlying subchondral bone with a scalpel. 5 mm diametercylindrical explants, ranging from 2 to 2.8 mm in thickness, cored outusing a dermal punch (Acuderm, Inc., Ft. Lauderdale, Fla.). Explantstaken from adjacent sites were used as matched controls for each groupin each experiment to minimize variation in cartilage response due todifferences in thickness and location within the joint. Each explant wasweighed and allowed to stabilize at 37° C. and 5% CO₂ for 48 hours inDulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetalbovine serum (FBS), L-glutamine, antibiotics and 50 mg/ml ascorbic acid.

Loading apparatus and procedure. An Instron 8511 servohydraulic testingmachine (Instron Corporation, Boston, Mass.) was used to mechanicallyload the cartilage explants. Explants were transported from theincubator to the Instron machine in sealed cell culture dishes. Thetotal time duration of loading for a single experiment varied fromapproximately 1 to 2 hours depending on the number of samples. Duringthis time, explants were maintained at 37° C. The explant wascentralized on a stainless steel loading platform and a radiallyunconfined compressive load was applied through an impermeable stainlesssteel platen. Explants were subjected to a single 500 msec trapezoidalloading waveform at the selected stress (14 MPa) or strain level (30%).This loading protocol was chosen to simulate an impact injury and hadbeen found to consistently produce between 20 and 40% apoptosis in pilottests with bovine cartilage samples. Control explants were placed in themachine but not loaded. Both control and loaded explants werere-cultured in fresh culture media immediately after loading. Stress andstrain data were recorded during the test.

Response to mechanical injury. To establish extent of tissue response tomechanical injury one stress level of 14 MPa was selected. Prior pilottests demonstrated that loads below 7 MPa did not result in anymeasurable cell death and loads above 20 MPa resulted in extensivestructural damage of the tissue. Eight explants each taken from threedonor tibial condyles were tested at this stress level. Explants takenfrom adjacent cartilage were selected as paired non-loaded controls tocontrol for differences in site of origin. Explants were examined 96hours after loading for apoptosis.

Time response. To establish the time response after injury, explantswere loaded at 30% strain and cultured for 3, 6, 12, 24, 48 and 96hours, and 24, 48, 96 and 168 hours after injury in separateexperiments. Due to the variation in thickness and material propertiesof cartilage taken from different locations within the same joint,strain controlled experiments were performed as they were found toproduce more consistent injury. Percent apoptosis and GAG release wasmeasured at the end of each culture period.

Caspase inhibition. To determine whether caspase inhibitors couldprevent apoptosis, explants were cultured in 100 mM z-VAD.fmk (a broadspectrum caspase inhibitor) immediately after injury (30% strain level).Percentage apoptosis was measured at 96 hours post-injury in threegroups (control, loaded, and loaded with z-VAD.fmk) with ten explantseach in two separate experiments.

Caspase inhibition. The presence of a time delay in the establishment ofapoptosis suggests potential for agents that may inhibit this responseafter mechanical injury. Although apoptosis can be induced by via twomajor pathways (via death receptors or mitochondria), overlaps exist inthe sequential activation of caspases 3, 6 and 7. Several studies havedemonstrated the efficacy of caspase inhibition in preventing apoptosisin a variety of settings. Mechanically induced apoptosis can also bereduced by caspase inhibition. Explants cultured in z-VAD.fmkdemonstrated a mean 50% reduction in apoptotic rates (p<0.05). Theseeffects of caspase inhibition support the above findings of apoptosisdetected by TUNEL, since cells undergoing necrosis cannot be rescued bycaspase inhibition. In addition, it suggests that cells that aretriggered to undergo apoptosis can be rescued, opening possibilities forenhancing cartilage repair by increasing or maintaining cell viability.

Glycosaminoglycan release assay. To measure glycosaminoglycan release,the concentration of sulfated glycosaminoglycans (GAG) in culture mediawas measured using 1,9-Dimethylene Blue as a monitor ofspectrophotometric changes which occur during the formation of thesulfated GAG dye complex. To generate a standard curve,chondroitin-6-sulfate (Sigma, St. Louis, Mo.) was used at concentrationsbetween 1 and 200 mg/ml. GAG release was expressed as percent GAGcontent of control explants.

Apoptosis detection. Explants loaded as described above were fixed in10% buffered formalin. In situ detection of apoptosis was performed on 5mm thick sections using MEBSTAIN Apoptosis Kit (MBL, Nagoya, Japan).This uses fluorescein-dUTP to label DNA strand breaks (TUNEL method) andhence allows direct detection of DNA fragmentation. Cells demonstratingapoptosis emit a bright green fluorescence while normal cells display anorange color due to propidium iodide counterstaining. Apoptosis wasquantified by counting the number of cells demonstrating apoptosis andwas expressed as a percentage of the total number of cells. For furtherconfirmation of apoptosis, several control and loaded explants weredivided into two halves. One half underwent fluorescein-dUTP labeling asdescribed above. The other half was fixed in 2.5% glutaraldehydebuffered with 0.1 M cacodylate (pH 7.2), rinsed in cacodylate buffer,postfixed for 1 hour in 2% OsO₄ buffered with cacodylate, dehydrated ina graded ethanol series, and embedded in Polybed 812 (Polysciences,Warrington, Pa.). Thin sections were stained with uranyl acetate andlead citrate and examined under electron microscopy for features ofapoptosis. In addition, representative histologic sections were examinedfor presence of a caspase cleavage site in cytokeratin 18 that exposes aneo-epitope specific for apoptosis. This was detected byimmunohistochemical staining with M30 (Cytodeath, Boehringer Mannheim,Eugene, Oreg.), a monoclonal antibody that recognizes the neo-epitopeexposed in early apoptosis.

Example 3 Additional Studies

Chondrocyte apoptosis after mechanical injury to bovine cartilage. Toaddress the form of cell death after mechanical injury, full thicknesscartilage explants from the normal weight-bearing portions of maturebovine femoral condyles were selected. Explants from adjacent sites wereused as matched controls for each group in each experiment to minimizevariation in cartilage response due to difference in thickness andlocation within the joint. An Instron 8511 servohydraulic testingmachine was used to mechanically load the cartilage explants. Toestablish extent of mechanical injury three stress levels were selected:low load (7 MPa), moderate load (14 MPa) and high load (23 MPa). Priortests had demonstrated that loads below 7 MPa did not result in anydetectable cell death and loads above 23 MPa resulted in extensivestructural damage of the tissue. In each experiment, four explants eachwere tested at the three stress levels. The range of maximum tissuecompression obtained with each loading level was mean 40% strain (range31-49%) for the low load group, mean 67% strain (range 44-77%) for themoderate load group and 83% strain (range 74-89%) for the high loadgroup. The explants were cultured for 48 h after loading and apoptosiswas assessed by TUNEL. The low load explants did not show anysignificant difference in apoptosis rate as compared to the unloadedcontrols. Both moderate and high load explants exhibited significantlyhigher apoptosis rates at 40% and 47%, respectively (p<0.05) (Table 2).

TABLE 2 Load-induced apoptosis in human, bovine and rabbit cartilageType of explant Source Ctr Load Full thickness cartilage Bovine femoral(n = 20) 7 (0.7) 43 (7.5) Full thickness cartilage Human femoral/ 11(3.1)  32 (9.6) tibial (n = 8) Full thickness cartilage Human tali (n =10) 12 (7.1)  26 (8.2) Osteochondral Rabbit patellae (n = 4) 1 (2.1) 15(4.3) Osteochondral Human patellae (n = 4) 3 (0.9) 17 (7.9) Fullthickness cartilage = 5 mm diameter explants Osteochondral = wholepatellaThe control explants demonstrated a small number of apoptotic cells,mainly located in the superficial zone. The high load explants containedapoptotic cells predominantly in the superficial and intermediate zonesin various stages of apoptosis, including chromatin condensation, cellshrinkage and the formation of blebs at the cell membrane. Associatedwith this is the accumulation of apoptotic bodies in the extracellularmatrix adjacent to apoptotic cells. A small percentage of cells withabnormal morphology (<5%) had necrotic appearance on electronmicroscopy.

Release of sulfated glycosaminoglycans (GAG) was analyzed as a measureof the extent of damage sustained by the explant. GAG release increasedwith intensity of loading within the range tested at 48 hours afterloading. There was no significant difference between the unloaded andthe low load samples. Moderate and high load explants releasedsignificantly higher levels of GAG than the unloaded controls (p<0.02).GAG depletion was assessed by dimethylene blue binding assays of theculture supernatants and this correlated with the reduced of safranin Ostaining of the cartilage sections.

The next set of experiments determined whether load-induced apoptosiswas sensitive to pharmacologic manipulation and whether inhibition ofapoptosis was associated with reduced GAG depletion. The broad-spectrumcaspase inhibitor z-VAD.fmk caused a >50% reduction in the apoptosisrate of the high and intermediate load groups (Table 3). Associated withthe reduction in apoptosis was a normalization of the GAG depletion.

TABLE 3 Pharmacologic modulation of apoptosis after cartilage injury invitro Loaded + Source Agent Control Loaded Agent Bovine z-VAD.fmk 16(±8.4) 65 (±13.2)* 31 (±13.9)* Bovine IGF-1 10 (±3.3) 61 (±12.8)* 42(±10.9)* Bovine Dexameth. Same as above Same as above 47 (±11.2)* Humanz-VAD.fmk  9 (±4.5) 44 (±9.9)*  34 (±10.2)* Human IGF-1  3 (±3.3) 17(±5.7)** 10 (±6.2)** Bovine (femoral condyles) and Human cartilage(femoral condyles and tibial plateaus) *Loaded at 23 MPa; **Loaded at30% strain Two loading protocols: Stress controlled (23 MPa); Straincontrolled (30% strain) Sample size: n = 8 to 10 in each group Mean(±SD) apoptosis rates in different groups

Intraarticular injection of caspase inhibitor in rabbits with ligamenttransection. A pilot study was performed to assess feasibility andefficacy of caspase inhibition in vivo. Eight New Zealand White rabbitswere divided into anterior cruciate ligament transection (ACLT) andACLT+caspase inhibitor (CI) groups. Both groups underwent bilateralACLT. The ACLT+CI group was treated with intra-articular injections of25 μg of z-VAD.fmk three times per week for six weeks while the controlgroup received saline injections. Rabbits were euthanised at six weeksand femoral and tibial articular cartilage evaluated by India inkstaining and histologic Mankin grading after Safranin-O stain.

Under India ink examination, all the ACLT rabbits demonstrated cartilagelesions on both femoral condyles, lateral tibial condyles andposteromedial tibial condyles, ranging from grade III (overtfibrillation) to grade IVa (erosions>5 mm). The ACLT+CI rabbitsdemonstrated lesions that were smaller in area. The grade ranged from II(minimum fibrillation), with only one rabbit (two knees) having a GradeIII to IVa lesion. Histologically, more knees from the ACLT group hadhigher Mankin scores. 87% (⅞) ACLT knees had grades 5 or higher, while50% ( 4/8) ACLT+CI knees were graded 5 or higher. The India ink stainedfemoral condyles were photographed and digital images were used toquantify the surface areas of the lesions. This revealed that caspaseinhibitor injection reduced the size of the cartilage lesions by ˜80%.

Chondrocyte apoptosis after mechanical injury to human cartilage. In anadditional study the apoptosis response of human articular cartilage tomechanical stress was examined. Full thickness human cartilage explants,5 mm in diameter were subjected to a single static mechanical stress of14 MPa for 500 msec under radially unconfined compression. GAG releaseand percentage of cells undergoing apoptosis were measured at 96 hoursafter injury. To establish the time course of apoptosis, explants weresubjected to 30% strain and cultured for varying intervals up to 7 daysafter injury. A group of loaded explants were also treated with thebroad spectrum caspase inhibitor z-VAD.fmk after injury. Meanchondrocyte apoptosis of 34% (SD±11) was observed at 96 hours inresponse to mechanical loading at 14 MPa, compared to 4% (SD±2) in thenon-loaded explants. GAG release was also higher for the loadedexplants, mean 1.9%, (SD±0.14) of total GAG content, compared to controlexplants, mean 0.8%, (SD±0.28). The percentage of apoptotic cells alsocorrelated with the level of GAG release into the culture media. Thepercentage of apoptotic chondrocytes demonstrated a progressive increasefrom 6 hours to 7 days post-injury. When loaded explants were culturedin z-VAD.fmk after injury, a 50% reduction in apoptosis rates was seen.Thus, the apoptosis response of human cartilage to mechanical stress issimilar to that seen with bovine tissue.

1. A process of inhibiting apoptotic cell death in cartilage followingtraumatic mechanical injury of the cartilage of a human patient,comprising (a) identifying a human patient having a traumatic mechanicalinjury to cartilage; and (b) inhibiting the activity ofcysteine-aspartate-specific proteases in the injured cartilage.
 2. Theprocess of claim 1, wherein the activity of cysteine-aspartate-specificproteases is inhibited by contacting the cartilage with a caspaseinhibitor.
 3. The process of claim 2, wherein the caspase inhibitor is afluoromethylketone caspase inhibitor.
 4. The process of claim 2, whereinthe caspase inhibitor is a broad spectrum caspase inhibitor.
 5. Theprocess of claim 2, wherein the caspase inhibitor is administered to thepatient within seven days of the injury.
 6. The process of claim 2,wherein the caspase inhibitor is administered in an amount that issufficient to achieve a concentration of from about 20 μM to about 250μM in an interstitial fluid which perfuses the injured cartilage.
 7. Theprocess of claim 1, wherein the traumatic mechanical injury of thecartilage is blunt traumatic mechanical injury.
 8. The process of claim1, wherein the traumatic mechanical injury of the cartilage is externaltraumatic mechanical injury.
 9. The process of claim 1, wherein thecartilage is articular cartilage.
 10. A process of inhibitingglycosaminoglycan release from cartilage following mechanical injury ofthe cartilage of a human patient, comprising inhibiting apoptotic celldeath in the injured cartilage by inhibiting the activity ofcysteine-aspartate-specific proteases in the injured cartilage.
 11. Theprocess of claim 10, wherein the activity of cysteine-aspartate-specificproteases is inhibited by contacting the cartilage with a caspaseinhibitor.
 12. The process of claim 11, wherein the caspase inhibitor isa fluoromethylketone caspase inhibitor.
 13. The process of claim 11,wherein the caspase inhibitor is a broad spectrum caspase inhibitor. 14.The process of claim 11, wherein the caspase inhibitor is administeredto the patient within seven days of the injury.
 15. The process of claim11, wherein the caspase inhibitor is administered in an amount that issufficient to achieve a concentration of from about 20 μM to about 250μM in an interstitial fluid which perfuses the injured cartilage. 16.The process of claim 10, wherein the traumatic mechanical injury of thecartilage is blunt traumatic mechanical injury.
 17. The process of claim10, wherein the traumatic mechanical injury of the cartilage is externaltraumatic mechanical injury.
 18. The process of claim 10, wherein thecartilage is articular cartilage.